Killer lymphocytes release their cytotoxic granule contents when they recognize cells infected with intracellular pathogens. Cytotoxic granule proteases (granzymes, Gzm), delivered into the target cell by the membrane perturbing protein perforin (PFN), trigger host cell apoptosis. However, what happens to intracellular bacteria during this process is unclear. Cytotoxic granules of humans and most other mammals, but not rodents, contain another pore-forming protein, granulysin (GNLY), which preferentially disrupts cholesterol-poor bacterial, fungal and parasite membranes, to lyse extracellular pathogens when applied at high concentrations. We recently found that GNLY delivers Gzms into intracellular and extracellular bacteria, where they rapidly kill bacteria. In aerobic bacteria, Gzs cleave bacterial electron transport chain (ETC) complex I components to disrupt electron transport and generate superoxide anion and at the same time cleave and disrupt oxidative stress defense enzymes that detoxify superoxide anion, rendering the bacteria defenseless. Anaerobic bacteria are also killed, but more slowly. The importance of this pathway in vivo was assessed using mice bearing a GNLY transgene (Tg), expressed, like the human protein, only in activated killer cells. GNLY-Tg mice cleared L. monocytogenes (Lm) much more effectively than wild-type (WT) mice. The goal of this application is to investigate the hypothesis that Gzms, GNLY and PFN in cytotoxic T cells and NK cells and other innate-like lymphocytes play an important role in bacterial immune defense and to define under what in vivo conditions it is important and the pathways that mediate bacterial cell death. Based on preliminary analysis of Gzm targets in bacteria, we hypothesize that the Gzms activate common programs of cell death in bacteria that disrupt key biosynthetic and metabolic pathways and stress responses. We will answer the following questions: Which types of lymphocytes express GNLY and do bacteria or bacterial products induce its expression? Are killer cells important against bacteria that do not use oxidative phosphorylation? Are extracellular bacteria targeted under some conditions? Is GNLY on its own used for NK and T cell-mediated elimination of extracellular bacteria, while all 3 effector molecules are employed to eliminate intracellular bacteria? Are there important bacterial substrates that contribute to bacterial elimination besides ETC complex I? We will answer these questions using a few model organisms. The in vivo consequences of GNLY and Gzm-dependent bacterial defense will be examined by comparing bacterial infections in WT and GNLY-Tg mice, focusing on the skin and gut, where bacteria enter the body. Our specific aims are to (1) define characteristics of bacterial death programs activated by Gzms and GNLY, (2) identify Gzm substrates and investigate whether and how the Gzms disrupt bacterial metabolism, biosynthesis and the stress response, and (3) determine the role of cytotoxic effector molecules (GNLY, Gzms, PFN) and classes of killer lymphocytes in protection from infection by diverse bacterial strains.